The present disclosure is related to fluorescence imaging and spectroscopy, in particular the use of fluorescence to detect and/or treat cancer by marking tumor cells with an appropriate fluorescing agent. One difficulty in using fluorescence for medical diagnostics and therapeutics is quantifying the fluorescence in tissue. Fluorescence signals are strongly affected by variations in the tissue absorption and transport scattering properties (i.e. tissue optical properties), whereas often the objective is to quantify the fluorescence based on fluorophore concentration alone.
Fluorescence measurement is of interest for applications such as photosensitizer dosimetry during photodynamic therapy (Finlay et al. 2006), fluorescence image-guided surgery (Stummer et al. 1998), detection of cancerous or dysplastic lesions (Muller et al., 2001) and in time kinetics studies of fluorescing drugs (Sroka et al. 1996). The shape and intensity of the fluorescence spectrum contain useful information on the identity and abundance of fluorophores in tissue. However, accuracy of quantitative fluorescence measurement is complicated by the distorting effects of light absorption and scattering by the tissue and the variations in measurement geometry (e.g. detector-to-tissue surface distance). Untangling these confounding effects is important for quantitative analysis of fluorescence.
Some methods have been developed in an attempt to diminish these distorting effects to better utilize fluorescence information. Many of these techniques use a diffuse reflectance signal to correct the fluorescence signal from optical properties variation. Wu and coworkers have developed a fluorescence photon migration model to produce a relation with the diffuse reflectance that can be exploited to extract the quantitative fluorescence in tissue (Wu et al. 1993). On a different tack, a single optical fiber may be used for both source and collection, the concept being that detectable fluorescence events occur so close to the fiberoptic tip that absorption and scattering effects are minimal, analogous to how these effects are minimal for very thin tissue sections (Diamond et al. 2003).
Empirical methods with similar themes have also been developed. The single fiber method was used in conjunction with an empirically-derived correction factor dependent on the optical properties at the emission wavelength to further compensate for high tissue attenuation in the prostate during PDT studies (Finlay et al. 2006). A fluorescence/reflectance ratio has been used to quantify fluorophore concentration, but with the fluorescence and reflectance measured at different source-collector distances (Weersink et al. 2001). In all of the above methods, the excitation source operates in the region of low tissue absorption, which invalidates their use in the UV-blue-green end of the spectrum (i.e. approximately from 350-575 nm), where a very large subset of fluorophores have fluorescence absorption maxima, such as porphyrins, background autofluorescence and a multitude of artificial fluorescent dyes.
Ex vivo extraction techniques have also been developed that are based on homogenizing the tissue and diluting the analyte to the point that effects due to optical scattering and absorption are negligible (Lilge et al. 1997). These procedures are relatively time-consuming and open to error due to tissue handling or cryofreezing for post-processing. It would be useful to provide an in situ fluorometric approach that has applicability to a wide variety of fluorophores and tissues.